1Department of Chemistry, Wright State University, 2Department of Neuroscience, Cell Biology, and Physiology, Wright State University
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Anders, C. B., Baker, J. D., Stahler, A. C., Williams, A. J., Sisco, J. N., Trefry, J. C., et al. Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles. J. Vis. Exp. (68), e4167, doi:10.3791/4167 (2012).
Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 μg ml-1 down to 10 ml of 198.7 μg ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 μg ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 μg ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.
1. Synthesis of Colloidal AgNPs
The reaction mechanism for the Creighton method (slightly modified, inexpensive)22 is described in great detail in the Supporting information of reference Pavel et.al together with the undesired hydrolysis side-reaction of NaBH4 at room temperature or higher.23
2. Characterization of Colloidal AgNPs
A Cary 50 UV-VIS-NIR spectrophotometer (Varian Inc.) and a LabRamHR 800 Raman system (Horiba Jobin Yvon, Inc.) equipped an Olympus BX41 confocal Raman microscope, were utilized for AgNP characterization. The Cary WinUV software, LabSpec v.5 and Origin 8.0 software were employed for the data collection and analysis.
Note: The acquisition parameters will have to be optimized for other instrumentation models.
Determination of Surface Plasmon Resonance of Colloidal AgNPs via UV-Vis Spectrophotometry
Purity Test of Colloidal AgNPs via Raman spectroscopy
Due to the time limitation of the video demonstration (10-15 min video) and the space limitation of the protocol text (maximum 3 pages), this experimental section will not be videotaped.
3. Size-selection and Concentration of Colloidal AgNPs via Tangential Flow Ultrafiltration (TFU)
A KrosFlo II Research filtering system (Spectrum Laboratories, Rancho Dominguez, CA) was used to limit the AgNP polydispersity and to concentrate them (Figure 2). The three steps of the TFU process were: (1) Size-selection of AgNPs and AgNP-aggregates of 50-nm in diameter and larger using a 50-nm MidiKros polysulfone module (460 cm2), 2) Size selection and concentration of AgNPs of 1-20 nm in diameter using a 100-kD MidiKros filter (200 cm2), and (3) Further volume reduction using a 100-kD MicroKros polysulfone filter (20 cm2) (Figure 3).
4. Quantification of Silver Amount in Colloidal AgNPs by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES)
Each colloidal sample was chemically digested and the amount of silver was quantified by ICP-OES using an A 710E spectrometer (Varian Inc.). A linear regression calibration curve for silver (Figure 4) was constructed using eight silver standards (0, 3, 7, 10, 15, 25, 50, and 100 μg L-1), which were prepared from a 10,000 μg ml-1 silver standard for trace metal analysis (Ultra Scientific).
5. Size Distribution of Colloidal AgNPs via Transmission Electron Microscopy (TEM)
A Phillips EM 208S TEM was used to visualize the colloidal AgNPs. Electron micrographs were captured using a high resolution Gatan Bioscan camera and analyzed in ImageJ software.24
6. Representative Results
Synthesis and Characterization of Colloidal AgNPs
Four liters of Creighton colloidal AgNPs were successfully synthesized using the setup displayed in Figure 1A. The final colloid had a characteristic golden yellow color (Figure 1B).22, 23 The UV-Vis absorption spectrum of this colloid had a typical sharp, symmetrical surface plasmon peak (SPR) at 394 nm (Figure 1C). The Raman spectrum of the original Creighton colloid and the final 100-kD retentate presented only three vibrational modes, namely the bending (1640 cm-1) and symmetric and asymmetric stretching modes of H2O (3245 cm-1 and 3390 cm-1, respectively) (Figure 1D).
TFU of Colloidal AgNPs
The TFU setup and the schematic of the 3-step TFU process are depicted in Figures 2 and 3, respectively. In step 1, a 50-nm filter (460 cm2) was utilized to size-select and to remove AgNPs and AgNP-aggregates of 50-nm diameter and larger from the original colloid (about 100 ml of 50-nm retentate). This step was also accompanied by a small volume reduction from 4 L of original colloid down to 3.9 L of 50-nm filtrate. No backwashing or flow disruption step was used. The largest volume reduction (i.e., water removal) was obtained in step 2, when the 50-nm filtrate was subsequently run through a 100-kD filter (200 cm2). The resulting 100-kD retentate had a total volume of 50 ml. Most of the synthesis byproducts and excess reagents were eliminated in this step through the water solvent (3.850 ml of 100-kD filtrate). Further, AgNP concentration was achieved by the addition of a third filtration step to the previously reported procedure.19 In this step 3, a 100-kD filter of a smaller surface area (20 cm2) reduced the 100-kD retentate volume to 4.0 ml. The TEM measurements will demonstrate that this final 100-kD retentate consists mostly of lowly aggregated AgNPs of 1-20 nm in diameter.
ICP-OES and TEM of Colloidal AgNPs
A linear regression calibration curve (Figure 4) for silver was constructed from eight standards (0, 3, 7, 10, 15, 25, 50, and 100 μg L-1). The amount of silver in each of the four representative colloidal samples was then determined from the ICP-OES calibration curve through extrapolation: original colloid (15.2 ppm, Figure 3A), 50-nm filtrate (14.1 ppm, Figure 3B), first 100-kD retentate (683.1 ppm, Figure 3C) and final 100-kD retentate (8,538.9 ppm, Figure 3D). The actual yield of 15.2 ppm is very close to the typical theoretical yield of 15.4 ppm for the Creighton reaction. The extreme concentration of AgNPs (4 ml of 8,538.9 ppm) was reflected by a dramatic change in color from golden yellow for the original colloid to dark brown for the final 100-kD retentate (Figure 3, insets of vial pictures). The quality of the filters was found to be critical to the TFU process, in particular to step 1. The final retentate concentrations ranged from 3,390.1 ppm to 9,333.3 ppm depending on the condition of the filters (heavily used versus brand new). If the membrane pores become compromised, AgNPs that have diameters less than 50-nm will also be retained and will subsequently decrease the overall amount of AgNPs that is collected in the filtrate. Optimization of the filtration process to include pressure monitoring and proper cleaning can increase the life span of the filters.
Representative TEM micrographs of the original Creighton colloid and the final 100-kD retentate (step 3) are shown in Figure 5A and 5C, respectively. In their unaggregated state, AgNPs appear as black round areas on a lighter grey background. Approximately 800 AgNPs were identified in the TEM micrographs of each of the two samples and were analyzed using the Image J software. One particle was defined by a complete and enclosed perimeter. An area threshold value was set at 1.0 nm2 according to the resolution of the TEM micrographs. The AgNP counts and area data were then exported into Microsoft Excel and the AgNP diameters were extrapolated. The average AgNP diameter in the original colloid and the final 100-kD retentate were determined to be 9.3 nm and 11.1 nm, respectively. The diameter measurements of the AgNPs were then exported to Origin 8.0 software and a TEM size histogram was constructed for each sample (Figure 5B and 5D).
Figure 1. A) Synthesis setup, B) Characteristic color, C) UV-Vis absorption spectrum, and D) Raman spectrum of Creighton colloidal AgNPs.
Figure 2. TFU experimental setup for A) steps 1 and 2: I) Reservoir containing Creighton colloidal AgNPs. II) Reservoir for filtrate collection. III) Y-junction in tubing. IV) Peristaltic pump head. V) Either 50-nm or 100-kD Midi Kros filter. B) step 3: I) Reservoir containing Creighton colloidal AgNPs. II) Reservoir for filtrate collection. III) 100-kD Micro Kros filter.
Figure 3. Flowchart depicting the TFU process. The blue-shaded boxes mark the colloidal suspensions of AgNPs collected for further analysis. Vial photographs show A) Original colloid batch, B) 50-nm filtrate collected after processing the original colloid through the 50-nm filter (460 cm2), C) first 100-kD retentate obtained after volume reduction using the 100-kD Midi Kros filter (200 cm2), and D) final 100-kD retentate resulting from the volume reduction using the 100-kD Micro Kros filter (20 cm2). The 100-kD filtrate looks like water.
Figure 4. ICP-OES linear calibration constructed using eight silver standards: 0, 3, 7, 10, 15, 25, 50, and 100 μg L-1.
Figure 5. TEM micrographs of A) original Creighton AgNPs and C) final 100-kD retentate (scale bar is 100 nm). TEM size histograms constructed by analyzing approximately 800 AgNPs for B) original Creighton AgNPs, and D) final 100-kD retentate. The inset in Figure 5B shows the expanded 41-75 nm size range for comparison purposes. Click here to view larger figure.
UV-Vis Absorption Spectrophotometry and Raman Spectroscopy of Colloidal AgNPs
It is well known that the number of surface plasmon resonance peaks in the absorption spectrum of a colloid decreases as the symmetry of the AgNPs increases. Additionally, AgNP aggregation leads to the appearance of broader or red-shifted peaks.25,26 The presence of a single, sharp and symmetrical SPR peak at 394 nm is indicative of small, spherical AgNPs of moderate aggregation and size distribution.
The purity of the colloidal samples before and after ultrafiltration was demonstrated by the Raman spectra of the original Creighton colloid and the final 100-kD retentate, which exhibited only three vibrational modes characteristic to H2O. The Raman signal associated with organic impurities or ultrafiltration contaminants of large Raman cross-sections would be enhanced through the immediate proximity to the AgNP surface (i.e., the so-called surface-enhanced Raman spectroscopy (SERS) effect).
ICP-OES and TEM of Ultrafiltered Colloidal AgNPs
The addition of a third, 100-kD filtration step to the previously reported TFU procedure19 facilitated the successful reduction of a larger volume of Creighton colloidal AgNPs (4L batch of 15.2 ppm) in a 1,000-fold smaller volume of retentate (4 ml of 8,538.9 ppm). This corresponds to a TFU concentration yield of approximately 62% taking into account the amount of AgNPs and AgNP-aggregates of 50-nm diameter and larger that were removed. The degree of concentration is remarkable because the final 100-kD retentate mostly consisted of monodisperse AgNPs that were 1-20 nm in diameter and free from excess reagents and byproducts. The third, 100-kD filtration step improved the concentration yield from 45%20 to 62%. Further TFU improvements in the size-selection and concentration of AgNPs could be obtained by utilizing additional hollow fiber membranes. Filters of pore size ranging from 1,000 kD to 10 kD and surface areas from 5.1 m2 to 8 cm2 are currently available for both hydrophobic and hydrophilic samples. Buffer exchange can also be performed during TFU, depending on downstream applications. When the volume reduction exceeds 800-fold (i.e., when the volume is reduced from 4 L to less than 5 ml), there is a decrease in the stability and shelf life of the colloidal suspension due to the extreme degree of concentration. The shelf life for these highly concentrated, unfunctionalized AgNP is approximately one to two weeks at 10 °C. While inconvenient, this limitation is managed through careful research planning and preparation. This extreme degree of concentration was desired for ongoing nanotoxicity studies at various concentrations. Less concentrated batches of AgNPs are expected to have better stability and longer shelf life.
Visual inspection of the TEM images (Figure 5A and 5C) showed an increased frequency of minimally aggregated AgNPs in the final 100-kD retentate as compared with the original colloid. The TEM size histograms of the two colloidal samples (Figure 5B and 5D) further confirmed that the polydispersity of the Creighton colloidal AgNPs was limited through TFU. Further polydispersity limitation may be achieved by employing a series of filtration membranes of smaller pore sizes. The diameters of the Creighton AgNPs ranged from 1 nm to 75 nm (Figure 5B and the inset showing the expanded 41-75 nm size bins), while the AgNPs and/or AgNP-aggregates of 50 nm and larger (0.9% out of percent total AgNPs) were absent in the TEM size histogram of the final 100-kD sample (Figure 5D). The 100-kD retentate was comprised mostly of AgNPs that had diameters of 1-20 nm; there was a small contribution (12.4%) from AgNPs in the 21-40 size bins. Figure 5B and 5D confirmed that the size distribution trend was retained for the 100-kD retentate during the TFU process with the exception of the 1-5 nm size range. There was a noticeable decrease in the frequency of the smaller AgNPs of 1-5 nm in diameter for the 100-kD sample (from 33.2% to 21.3%), which was attributed to AgNP passage through the membrane filter into the filtrate. As a result, the average AgNP diameter increased from 9.3 nm for the original colloid to 11.1 nm for the final 100-kD retentate. Because approximately 800 AgNPs were analyzed for both colloidal samples, the decreased frequency of smaller AgNPs in the 1-5 nm (11.9%) and 6-10 nm (1.3%) size ranges was accompanied by a corresponding increase in the frequency of larger AgNPs in the 11-25 nm size bins (i.e., about 12.8% from the original colloid to the 100-kD retentate).
In conclusion, TFU proved to be an efficient, "green" method for the size-selection and concentration of colloidal AgNPs with minimal aggregation at various volume scales. Eliminating the use of chemically aggressive reagents or organic solvents from the AgNP synthesis (for better size, shape, and aggregation control) may significantly reduce AgNP toxicity while improving their therapeutic index. AgNPs of limited polydispersity may find other immediate industrial and research applications due to their improved catalytic,27 optoelectronic28, 29 or SERS-based biosensing properties.9,19,30,3131 A very recent study by Lander et al.32 showed that micro and ultrafiltration membranes made of five different polymeric materials (polysulfone, polyethersulfone, nylon, cellulose acetate, and polyvinylidenefluoride) may be successfully implemented for the size selection of functionalized NPs. These NPs of 2-10 nm in diameter had Ag, Au or Ti2O cores and were functionalized with organic polymer coatings that led to positive or negative surface charges. Both the cores and the surface functionality of the NPs were found to play a major role in the NP retention or passage through the membranes (0.2 nm to 0.22 μm). As expected, the positively charged NPs were entirely rejected (> 99%) by the negatively charged membranes that had 20-times larger pore size than the NP diameters. From these experiments, one learns that the interaction mechanism should be carefully considered in future studies with functionalized NPs.
No conflicts of interest declared.
Funding from the National Science Foundation through the NUE in Engineering and the LEADER Consortium Programs is gratefully acknowledged.
|Silver nitrate (AgNO3)||Acros Organics Inc.||CAS: 7761-88-8|
|Sodium borohydride (NaBH4)||Acros Organics Inc.||CAS: 16940-66-2|
|Nitric acid (HNO3, Optima)||Fisher Scientific Inc.||A467-1||Trace metal grade for ICP analysis|
|10,000 μg ml-1 silver standard, EnviroConcentrate||Ultra Scientific||US-IAA-047|
|KrosFlo Research IIi Tangential Flow Filtration System||Spectrum Laboratories Inc.||SYR2-U20-01N|
|0.05 μm PS (0.5 mm) 460 cm2||Spectrum Laboratories Inc.||X30S-900-02N|
|Midi 100 kD PS 200 cm2||Spectrum Laboratories Inc.||X3-100S-901-02N|
|Micro100 kD PS 20 cm2||Spectrum Laboratories Inc.||X1AB-300-10N|
|MasterFlex C-Flex tubing L/S Size 17||Cole-Palmer Instrument Co.||06424-17|
|MasterFlex C-Flex tubing L/S Size 14||Cole-Palmer Instrument Co.||06424-14|
|Cary 50 UV-VIS-NIR spectrophotometer||Varian Inc.|
|LabRam HR 800 system||Horiba Jobin Yvon Inc.|
|Varian 710ES ICP-–S||Varian Inc.|
Table 1. Specific reagents and equipment.